In many applications ranging from medical analysis to environmental detection, the precise delivery of a liquid of known but varying composition is important. The delivery of such a liquid at low flow rates is useful in some applications such as those involving capillary separation. One field of application is liquid chromatography.
In liquid chromatography, chemical separations may be performed by flowing a fluid (the mobile phase) past an immobilized material (the stationary phase) inside a liquid chromatography (LC) column. This technique is used for chemical analysis by injecting a sample consisting of multiple components into one end of the LC column, allowing them to be separated into distinct bands as the sample flows through the LC column, and detecting those bands near the exit end of the LC column. In those systems, the separation is governed by the dynamic partitioning of the analyte between the mobile phase and the stationary phase. Control of the separation may be achieved by adjusting the composition of the mobile phase or the stationary phase or both to influence analyte partitioning.
High-performance liquid chromatography (HPLC), which is an established analytical technique, relies on high-pressure mechanical pumps to flow the mobile phase through the LC columns that are packed with immobilized particles. The stationary phase may comprise the particles themselves or particles with a chemical layer bonded to them. "Isocratic" separations are achieved by using a single pump to flow a mobile phase of constant composition through the packed LC column.
A. Capillary Analytical Methods
Several analytical methods using miniaturized or capillary columns have been developed, including capillary zone electrophoresis (CZE) and micro-HPLC. The CZE technique, in which a voltage potential is applied to a buffer-filled capillary to generate electro-osmotic flow, provides excellent efficiency in separating charged species via their different electrophoretic mobilities. In CZE, the capillary is typically made of silica, a material that forms fixed negative charges on the inner capillary wall in the presence of a solution of the correct pH containing electrolytes. Before the voltage gradient is applied, cations in the electrolyte solution will be attracted to these fixed negative charges, forming a so-called double layer at the capillary wall. Application of the voltage gradient creates a net movement of the cations loosely associated with the fixed negative charges at the electrolyte/silica interface. This movement, referred to as electro-osmotic flow, causes the bulk of the electrolyte solution to be dragged toward the negatively charged discharge outlet. A key disadvantage of the CZE approach, however, is that it cannot be used to resolve neutral compounds.
Micro-HPLC, on the other hand, employs a stationary phase material in a capillary column and provides high selectivity in a wide range of applications because of the variety of stationary phase materials available for HPLC. The column efficiency, however, is reduced in micro-HPLC because the mobile phase is driven through the capillary separation column using high mechanical pump pressure, which results in a parabolic flow velocity profile.
The emerging liquid chromatography technique known as capillary electrochromatography (CEC) combines the high selectivity of micro-HPLC and the high efficiency of CZE. In CEC, a capillary column is packed with a stationary phase material similar to that used in micro-HPLC. The mobile phase, however, is pumped through the capillary column using an applied electric field to create an electro-osmotic flow, similar to that in CZE, rather than using high pressure mechanical pumps. The CEC approach can thus achieve the high efficiency of CZE. In addition, as in the case with micro-HPLC, CEC may be used to analyze neutral compounds that are not separable by CZE. The miniaturization of the separation column by using a capillary column in CEC offers several advantages, including improved efficiency, mass detection sensitivity, low solvent consumption, small sample quantity, and easier coupling to detector such as mass spectrometers and flame-based detectors.
B. Gradient Elution
Gradient elution is a process by which the mobile phase composition is varied during separation for separating a wide variety of complex samples. The process has been developed for HPLC and micro-HPLC. The gradient elution approach is useful when the components of the mixture have a range of properties and no single mobile phase composition is appropriate for separating all of them.
In HPLC, the creation of the solvent gradient is accomplished by using two high-pressure mechanical pumps to deliver two different fluids into a small mixing chamber. The composition of the mobile phase is controlled and varied by adjusting the relative output flows from the individual pumps to achieve gradient elution.
In chemical analysis using electrokinetic techniques, such as CZE or CEC, current approaches include the use of mechanical means for gradient elution, such as the use of a pump or manual addition to a reservoir to deliver a gradient to a capillary column, or an electric field to deliver ionic species via electrophoresis to vary the composition in a separation column. For instance, one system uses a pressure-driven HPLC gradient system to deliver a solvent gradient to a packed capillary LC separation column. An applied electric potential is combined with pressure to perform the chromatographic separation. This technique is known as "pseudo-electrochromatography" or "pressure-assisted CEC." The key disadvantages of this method include slow response and low reliability at low flow rates.
Others have used different mechanical means for changing the mobile phase composition in a separation column during separation. A syringe-type doser has been used to pump the modifying electrolyte into the background electrolyte chamber to form pH gradients. Some have used a programmed solvent-delivery system and a split injector to generate pH gradients. Still others have proposed an HPLC gradient system to generate pH gradients and flow gradients in CZE. A stepwise gradient in micellar electrokinetic capillary chromatography (MECC) has been produced by manually pipetting aliquots of a gradient solvent containing 2-propanol into the inlet reservoir of the capillary. These systems have similar drawbacks as those of the pressure-assisted CEC system.
Another type of technique uses electric fields to generate pH gradients in isotachophoresis and capillary electrophoresis (CE). One such system employs two buffer chambers, each with its own electrode, to cause the migration of two different ionic species into the capillary during separation. The two buffer reservoirs are separated from the capillary by semipermeable membranes which allows ionic species to pass therethrough. This technique is appropriate for modifying the ionic content in a separation column. It cannot, however, be used for delivering an arbitrary flow.
Low volume flow rates of varying composition are required in capillary-based separation techniques when a single mobile phase is insufficient to separate all of the chemical components of a sample. Prior chromatography systems using miniature columns either are unable to employ gradient elution or use mechanical delivery means which produces gradients with parabolic flow velocity profiles and loss of separation efficiency. Conventional approaches of providing a variable composition mobile phase at low flow rates are expensive to implement, slow to respond to external control, and are generally unreliable in composition for flow rates of less than 1 .mu.L/min. Although the miniaturization of separation columns in CEC offers the above-mentioned advantages, delivering a .mu.L/min gradient flow into a capillary column (e.g., 10-100 .mu.m i.d.) packed with micrometer-size particles poses a difficult problem. There is therefore a need for a liquid delivery system for controlling precisely the delivery and the composition of a liquid having varying composition at sub-.mu.L/min flow rates.